The present invention relates to a method and apparatus for the photometric determination of biological agglutination using light scattering. More particularly, the present invention relates to a method and apparatus for the photometric determination of biological agglutination of test samples by detecting scattered light through the test samples using a laser beam source and at least one photodetector.
The importance of determining biological agglutination systems such as antigens, antibodies, blood clotting, etc. with precision has long been recognized in various fields of life sciences as well as in the field of the therapy, and there has been stronger desire for the development of quantitative determination of antigens, antibodies, blood clotting, etc. with high precision as demand therefor has increased in recent years.
Determination heretofore prevailing which is a plate immune diffusion takes one or a few days and the judgement of the results obtained by this method is very complicated and requires skill. From this it follows that the judgement varies from person to person. This is one of the major defects of the conventional method.
Recently, there has been proposed a nephelometric immunoassay in order to detect the formation of antigen-antibody complexes with light scattering, thus improving operability and precision in quantitative determination. However, the conventional nephelometric immunoassay practically takes a relatively long time ranging from several tens of minutes to several hours and therefore is unsatisfactory when urgent detection or high speed treatment of a large amount of test samples is needed.
Further, it has heretofore been known to detect antigens or antibodies by reacting latex particles on which an antigen or an antibody is supported with a corresponding antibody or antigen on a glass plate and observing visually the state of agglutination which occurs. This method is disadvantageous since it gives only qualitative data by judging whether or not agglutination occurs on the glass plate and the judgement as to whether or not agglutination occurred will be apt to vary depending on personal conditions of those who carry out the detection in the borderline regions. A further disadvantage is that with the above method it is necessary to prepare a series of seriously diluted samples and judgement must be made on each diluted sample, which not only requires a lot of time and effort but also gives rise to semiquantitative data only, thus achieving but low precision.
On the other hand, it has been known to measure optical absorbance of a test sample in a reactor with a light whose wavelength falls in the near-infrared region in order to qualitatively determine the agglutination state. This method is disadvantageous in that on one hand, most of the transmission light to be detected is blank information which is ascribable to reduction by latex particles in a non-agglutinated state, and the change in the transmission light ascribable to the minor part of the latex particles which agglutinated is very small as compared with the blank value, and on the other hand, when measuring transmission, light sensitivity is lowered further because of multiple scattering in the light path since test samples has a very high turbidity and also it tends to be influenced by movement of the particles in the light path.
In the case where scattered light is utilized, a conventional method using an integrating-sphere photometer is also disadvantageous since the background value is large relative to the value due to agglutination as in the case of measuring the optical absorbance, resulting in decreases in sensitivity and stability.
As stated above, the conventional methods for measuring agglutination reactions are not satisfactory with respect to their precision since they are difficult to carry out in a stable manner with high sensitivity.
On the other hand, blood coagulation tests are extremely important and useful for successful therapy of a bleeding disorder in the patient or for the followup manangement of patients receiving therapy using an anticoagulant. Also, these tests must be done before an operation can be initiated.
The coagulation tests include measurements of prothrombin time hereafter referred to as "PT" and of activated partial thromboplastin time hereafter referred to as "APTT" which are well known and effected as measurements of an extrinsic blood clotting mechanism and intrinsic blood clotting mechanism, respectively.
Further, at present various automatic clottage detection devices capable of measuring PT and APTT are available commercially.
The process of blood coagulation is said to be an extremely complex chain reaction and involves blood coagulation factors I to XIII. It is therefore necessary to carry out quantitative determination of blood factors to see which factors are short and to what extent they are short in the case where disorder in PT or APTT is found.
For determining blood factors, a method has heretofore been used in which corrective reagents is used in PT or APTT tests to qualitatively detect which factor is insufficient and then a calibration curve should be obtained by the PT or APTT test using an appropriate blood factor-lacking plasma. This conventional method requires very complicated procedures and much work. In addition, the quantitative determination of the results is not so accurate since the determination method is indirect in nature.
In contrast to tests based on the gross blood coagulation, direct determination of each blood factor has recently been proposed which involves measurement of immune activity using an antigen-antibody reaction of measurement of biological activities using an enzymatic reaction with a synthetic substrate.
According to this method direct measurement can be carried out with ease and high specificity and precision. However, conventional apparatus for the measurement of PT or APTT cannot be used for this method and it is therefore necessary to provide therewith new costly devices so that the apparatus can be adapted to the direct measurement method described above.
On the contrary, conventional apparatus adapted for the direct determination of blood factors by the above method cannot be used for carrying out screening tests which are very useful in blood coagulating tests such as PT, APTT, etc.